Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

• This invention relates to an anti human pancreatic a-amylase monoclonal antibody (hereinafter referred to as . "anti P-Amy monoclonal antibody”), a quantitative determination of a human pancreatic a-amylase (hereinafter referred to as "P-Amy”), and a method for separation of P-Amy and a human salivary glands a-amylase (hereinafter referred to as "S-Amy”) and a reagent useful for the separation.
• anti P-Amy monoclonal antibody a quantitative determination of a human pancreatic a-amylase
• S-Amy a method for separation of P-Amy and a human salivary glands a-amylase
• a-amylase includes two kinds of isozymes, i.e. P-Amy and S-Amy, and correct diagnosis is difficult by merely measuring total a-amylase activity, and hence, the above two isozymes are usually subjected to differential quantitative determination.
• Conventional differential quantitative determination methods are, for example, electrophoretic method and inhibitor method.
• the electrophoretic method comprises applying the test samples to various carriers, subjecting to electrophoresis, staining the thus separated isozymes, and then measuring color density to know the activity of P-Amy and S-Amy. According to this method, however, the separation of P-Amy and S-Amy can not completely be done and hence precise differential determination can not be done. Moreover, this method requires much longer time in the whole procedure and hence is inconvenient for testing a large number of samples.
• the inhibitor method is carried out by using an S-Amy inhibitor of wheat origin, and the ratio of the activity of P-Amy to that of S-Amy is calculated from difference of the degree of inhibition.
• this method has low degree of specific inhibition to S-Amy and hence precise determination can not be done.
• this method has much variation among different test lots, and hence, a standard calibration curve must be drawn, which is very troublesome.
• An object of this invention is to provide anti P-Amy monoclonal antibody which has a high specificity and is useful for the differential quantitative determination of P-Amy and S-Amy.
• Another object of this invention is to provide a method for the quantitative determination of P-Amy and a method for the differential quantitative determination of P-Amy and S-Amy.
• a further object of this invention is to provide a method for the separation of P-Amy and S-Amy and also a kit useful therefor.
• the monoclonal antibody of this invention can be prepared by cell fusion method. More particularly, the monoclonal antibody of this invention can be prepared by the following steps (1) to (4):
• B cells are collected from the spleen of an animal which is well immunized with an antigen P-Amy.
• the antigen P-Amy is most preferably highly purified and is used in a large amount, but P-Amy having not so high purity may be used.
• a partially , purified human pancreatic juice is used as an antigen.
• a partial purification of P-Amy from human pancreatic juice can be done, for instance, by fractionation with ammonium sulfate.
• Immunization of an animal can be carried out by administering the antigen P-Amy to a mammal such as mouse or rat.
• Conditions for immunization such as amount of the antigen P-Amy, injection site, kinds and amount of the adjuvant, etc. may be the same as those conventionally used for production of antiserum.
• the immunization is usually carried out by administering parenterally an emulsion containing P-Amy and Freund's complete adjuvant to a mammal (e.g. mouse), followed by additional immunization in the same manner as above at an interval of about one week.
• the additional immunization is usually carried out 4 to 7 times. Three to five days after the final immunization, the spleen is taken out from the mammal and B cells are collected therefrom.
• the fusion is carried out by suspending the above B cells and known myeloma cells in a known serum medium for cell fusion in the presence.of a fusion accelerator and mixing them.
• the myloma cells are preferably originated from the same animal as that used for immunization in the above step (1) and do not grow in a medium used for selection of hybridoma and do not secrete any antibody by themselves.
• Such myeloma cells include, for example, a commercially available mouse myeloma cell line P3-X63-Ag8-Ul or equivalents thereof.
• Both cells are usually used in a ratio of 1 to 20 of B cells to 1 of myeloma cells.
• the fusion accelerator includes, for example, polyethylene glycol which has preferably a molecular weight of 1,000 to 7,500.
• the cultivation of fused cells is carried out by washing out the fusion accelerator therefrom, putting 0.1 - 0.2 ml of a suspension of the hybridoma in a medium for myeloma cells into each well on a 96-well titer plate (hereinafter optionally referred to merely as "well") and then incubating at about 37°C in 5 % carbon dioxide - air.
• a known medium for selection of hybridoma such as HAT medium is added to the medium with gradually increasing the addition ratio of the medium.
• HAT medium a medium for selection of hybridoma
• the medium for selection of hybridoma is exchanged with a medium for myeloma cells, i.e. a medium for hybridoma.
• the screening of the desired hybridoma can be carried out by checking the antibody titer and degree of cross reaction. That is, P-Amy or S-Amy and an insolubilized second antibody as mentioned hereinafter are added to a part of the culture medium, and the mixture is incubated, , centrifuged, and then the enzymatic activity of the resulting pellet is measured by a method as mentioned hereinafter, by which the antibody titer and the degree of cross reaction of P-Amy and S-Amy can be measured.
• the screened hybridoma is cloned by a limited dilution method and thereby there can be obtained the desired hybridoma which can substantially recognize only P-Amy.
• the hybridoma thus obtained can semi-permanently be kept by successive culture or in frozen state.
• the hybridoma obtained in the above step is cultured in vitro or in vivo, by which the desired monoclonal antibody can be obtained.
• the in vitro cultivation can be carried out by firstly cultivating several hybridoma on the 96-well titer plate, followed by cultivating with gradually increasing the scale.
• the in vivo cultivation can be carried out by intraperitoneal inoculation of the hybridoma to a mouse which is previously treated with pristane in order to make the growth of the fused cells easier. Ten to 20 days after the inoculation, a peritoneal fluid containing the desired monoclonal antibody is accumulated.
• the in vitro cultivation is done for producing a monoclonal antibody having high purity
• the in vivo cultivation is done in case of producing the monoclonal antibody in a large amount.
• the anti P-Amy monoclonal antibody can usually be obtained in an amount of 10 to 100 mg per one mouse.
• the peritoneal fluid obtained by the in vivo cultivation can be used as it stands, but it may be further purified when necessary.
• the separation and purification of monoclonal antibody from the in vitro culture medium or from the in vivo culture peritoneal fluid is optionally carried out by conventional physicochemical methods, for example, by an appropriate combination of salting out, centrifugation, dialysis, various column chromatographys such as affinity chromatography using protein A bound to an insoluble carrier, and the like.
• the anti P-Amy monoclonal antibody thus obtained can differentiate P-Amy and S-Amy. Besides, even after reacting immunologically with the monoclonal antibody of this invention, the P-Amy can decompose various kinds of substrates having various molecular weights, such as p-nitrophenylmaltoheptoside (hereinafter referred to as "PNP") (Boehringer Mannheim GmbH) which has a low molecular weight, or an insoluble blue starch polymer (Daiichi Kagaku Yakuhin K.K.) which has a high molecular weight.
• PNP p-nitrophenylmaltoheptoside
• the monoclonal antibody of this invention is useful as a reagent for differential quantitative determination of P-Amy and S-Amy.
• the quantitative determination of P-Amy can be done by the procedure comprising the following steps:
• the activity of P-Amy can directly be measured.
• the differential quantitative determination of P-Amy and S-Amy can be done by measuring the activity of a-amylase in the antigen-antibody complex and other materials (usually supernatant) as separated in the above-mentioned step (ii), or alternatively, by subtracting the activity of P-Amy from the activity of total a-amylase which is independently measured.
• the anti P-Amy monoclonal antibody in the step (i) is usually used in an excess amount over the amount corresponding to P-Amy contained in the test sample.
• the step (ii) can be carried out by a technique used for B/F separation in enzyme immunoassay. For instance, an insolubilized anti P-Amy monoclonal antibody is used instead of the anti P-Amy monoclonal antibody in the step (i), and after the immuno- reaction, the antigen-antibody complex is separated by centrifugation, or an insolubilized antibody against the antigen-antibody complex, i.e.
• an insolubilized second antibody is further reacted in the step (ii) and the resulting complex is separated by centrifugation.
• the insolubilization of antibody can be done by binding the anti P-Amy monoclonal antibody or the second antibody to an insoluble carrier by a conventional method.
• the insoluble carrier includes all conventional carriers, preferably bacterial cell wall debris as disclosed in U.S. Patent 4,166,767.
• the step (iii) and the measurement of total a-amylase can be done by known methods, for example by using a commercially available kit for quantitative determination of a-amylase.
• the kit includes Autopack a-amylase kit (sold by Boehringer Mannheim - Yamanouchi). In this kit, a solution containing PNP and a-glucosidase (hereinafter referred to as "PNP reagent”) is used as a substrate.
• the reagent used for the separation of P-Amy and S-Amy can be used in combinations with any commercially available kits for the quantitative determination of a-amylase.
• the reagent of this invention used for the separation of P-Amy and S-Amy contains at least
• phosphate buffer and bovine serum albumin are abbreviated as PBS and BSA, respectively.
• the emulsion (0.5 ml) is intraperitoneally administered to BALB/c mouse (Shizuoka Jikken Dobutsu Kyodo Kumiai), and after one week, the animal is immunized again in the same way. Further after one week, additional immunization (intravenous injection) is carried out. Thereafter, additional immunizations are repeated 6 times likewise. Three days after the final immunization, the spleen is taken out and B cells are collected therefrom.
• Mouse myeloma cells P3-X63-Ag8-U1 (ATCC catalogue No. CRL 1597) (1 x 10 7 cells) in logarithmic growth phase and spleen cells (1 x 10 8 cells) are mixed, and the mixture is washed with the medium I by centrifugation (400 x g, 10 minutes) and thereto is added slowly (in one minute) 50 % polyethylene glycol 1500 (Wako Junyaku Kogyo K.K.) (1 ml) which is kept at 37°C, and the mixture is stirred slowly. After one minute, the medium I (1 ml) which is kept at 37°C is added to the mixture likewise.
• each one ml of the medium I (20 ml) is added at an interval of 30 seconds, and the mixture is centrifuged (400 x g, 10 minutes) and the supernatant is removed. To the resulting pellets is added gradually the medium II (12 ml).
• Each 0.1 ml of the mixture is distributed into every well of the 96-well titer plate and subjected to cultivation at 37°C in 5 % carbon dioxide - air. After 20 hours, 0.1 ml of the medium III is added to each well. On 2nd, 3rd, 5th, 8th, 11th, 13th and 15th days (7 times in total), the old medium (0.1 ml) is removed, and a fresh medium III (0.1 ml) is added.
• the antibody titer and cross reaction of the culture liquid after 15 days cultivation are determined as follows.
• Pancreatic juice (250 ⁇ l, 92,000 IU/liter) is diluted with the following Buffer A to make a final volume of 50 ml.
• a buffer solution consisting of 0.1 % BSA - 0.9 % NaCl - 0.04 M PBS (pH 7.0)
• the antibody titer is determined in the following manner.
• the culture medium of hydridoma (50 ⁇ l), P-Amy solution (25 ⁇ l) and the suspension of insolubilized second antibody (50 ⁇ l) are mixed, and the mixture is incubated at 37°C for 30 minutes, and thereto is added physiological saline solution (4 ml).
• the mixture is centrifuged (3000 x g, 5 minutes), and the supernatant is removed.
• PNP reagent (1 ml), and the mixture is stirred and then incubated at 37°C.
• reaction terminator solution [0.1 M PBS (pH 11), 2 ml] is added thereto, and the reaction mixture is centrifuged, and the absorbance (405 nm) of the supernatant is measured. The well giving a higher absorbance is evaluated to contain an anti P-Amy monoclonal antibody having a higher antibody titer.
• the degree of cross reaction is investigated as to the culture medium having a . high antibody titer.
• the degree of cross reaction is determined.
• the test sample having a lower absorbance at 405 nm is evaluated to have a lower cross reactivity.
• a strain being capable of producing anti P-Amy monoclonal antibody having particularly high antibody titer and low cross reactivity is selected, and the hybridoma cells are diluted with the medium II so as to give a concentration of one cell per well on the 96-well titer plate and then cultivated. By repeating the above limited dilution, the cloning is achieved to give the desired hybridoma.
• the hybridoma can be kept in frozen state or by successive culture.
• BALB/c mouse which is previously intraperitoneally administered with pristane (0.5 ml) is intraperitoneally inoculated with the hybridoma (1 x 10 7 cells) obtained as above. After 15 days, there is obtained a peritoneal fluid (3 ml/mouse) containing the desired monoclonal antibody. To the peritoneal fluid is added an equivolume of saturated ammonium sulfate solution, and the mixture is stirred at 4°C for 30 minutes and centrifuged (5000 x g, 30 minutes), and the supernatant is removed. The pellet is dissolved in 0.05 M PBS (pH 7.4) - 0.14 M sodium chloride solution. This solution is dialyzed at 4°C overnight, centrifuged (5000 x g, 30 minutes), and the resulting supernatant is lyophilized to give crude monoclonal antibody.
• PBS pH 7.4
• the monoclonal antibody belongs to IgG1 subclass and the L chain thereof belongs to kappa.
• test sample is prepared by diluting a solution containing an equal amount of P-Amy and S-Amy by a two fold dilution method, and it is subjected to the differential quantitative determination in the following manner.
• test sample 25 ⁇ l
• an anti P-Amy monoclonal antibody which is prepared by diluting the peritoneal fluid obtained in Example 1-(2) in 1,000-fold with Buffer A] (50 ul)
• a suspension of insolubilized second antibody 50 ⁇ l
• physiological saline solution 4 ml
• the S-Amy activity is calculated by subtracting the P-Amy activity from total a-amylase activity.
• test sample 40 ⁇ l
• a suspension 150 ⁇ l of an insolubilized anti P-Amy monoclonal antibody which is prepared in the same manner as described in the item of Detection of antibody titer hereinbefore, and the mixture is incubated at 37°C for 10 minutes and then centrifuged (3000 x g, 5 minutes).
• the supernatant 25 ⁇ l is taken out and thereto is added PNP reagent (1 ml), and the mixture is incubated at 37°C for 30 minutes, and thereafter is added a reaction terminator (2 ml), and the absorbance at 405 nm is measured in a cuvette (light path: 1 cm).
• the P-Amy in the test sample is removed from the system by the insolubilized anti P-Amy monoclonal antibody, and hence, as is shown in Table 2, in the test samples (1), (4) and (7) which contain only P-Amy, the (b) value (corresponding to S-Amy activity) is all approximately zero. On the other hand, since S-Amy is not removed from the system, in the test samples (2), (5) and (8) which contain only S-Amy, the (b) value (corresponding to S-Amy activity) is approximately equal to the (a) value (total a-amylse activity). Besides, as is shown in Table 2, the mixing ratio of P-Amy and S-Amy [P/(P+S)] is approximately consistent to the b/a ratio. Thus, the method for differential quantitative determination of this invention is very excellent.
• the anti P-Amy monoclonal antibody of this invention is useful for the differential quantitative determination of P-Amy and S-Amy.
• P-Amy and S-Amy in human body fluids such as serum is differentially determined, it can be used for diagnosis of various diseases, particularly pancreatic disease.

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• 1986
  • 1986-05-13 JP JP61110233A patent/JPS6270399A/ja active Granted
  • 1986-05-15 EP EP19860903550 patent/EP0222923A1/de not_active Withdrawn
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